Modulation of secondary metabolite production by zinc binuclear...

ABSTRACT

Disclosed are methods for the modulation of secondary metabolite production by fungi through genetic manipulation of such fungi. Also disclosed are new commercial processes using ZBC proteins, or variants thereof, to significantly increase useful secondary metabolite production. Generally, the methods according to the invention comprise expressing in a fungus a ZBC protein or a variant thereof.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to secondary metabolite production byfungi. More particularly, the invention relates to modulation ofsecondary metabolite production by fungi through genetic manipulation ofsuch fungi.

[0003] 1. Description of the Related Art

[0004] Secondary metabolite production by various fungi has been anextremely important source of a variety of therapeutically significantpharmaceuticals. β-lactam antibacterials such as penicillin andcephalosporin are produced by Penicillium chrysogenum and Acremoniumcirysogenum, respectively, and these compounds are by far the mostfrequently used antibacterials (reviewed in Luengo and Penalva (1994),Prog. Ind. Microbiol. 29: 603-38; Jensen and Demain (1995),Biotechnology 28: 239-68; Brakhage (1998), Microbiol. Mol. Biol. Rev.62: 547-85). Cyclosporin A, a member of a class of cyclicundecapeptides, is produced by Tolypocladium inflatum. Cyclosporin Adramatically reduces morbidity and increases survival rates intransplant patients (Borel (1986), Prog. Allergy 38: 9-18). In addition,several fungal secondary metabolites are cholesterol lowering drugs,including lovastatin, which is made by Aspergillus terreus and severalother fungi (Alberts et al. (1980), Proc. Natl. Acad. Sci. USA 77:3957-3961). These and many other fungal secondary metabolites havecontributed greatly to health care throughout the world (see Demain(1992), Ciba Found. Symp. 171: 3-16; Bentley (1991), Crit. Rev.Biotechnol. 19: 1-40).

[0005] Unfortunately, many challenges are encountered between thedetection of a secondary metabolite activity to production ofsignificant quantities of pure drug. Thus, efforts have been made toimprove the production of secondary metabolites by fungi. Some of theseefforts have attempted to improve production by modification of thegrowth medium or the bioreactor used to carry out the fermentation.Buckland et al. (1989), in Topics in Industrial Microbiology: NovelMicrobial Products for Medicine and Agriculture, Elsevier, Amsterdam,pp. 161-169, discloses improved lovastatin production by modification ofcarbon source and also teaches the superiority of a hydrofoil axial-flowimpeller in the bioreactor. Other efforts have involved strainimprovements, either through re-isolation or random mutagenesis. Agathoset al. (1986), J. Ind. Microbiol. 1: 39-48, teaches that strainimprovement and process development together resulted in a ten-foldincrease in cyclosporin A production. While important, studies of thesetypes have still left much room for improvement in the production ofsecondary metabolites.

[0006] More recently, strains have been improved by manipulation of thegenes encoding the biosynthetic enzymes that catalyze the reactionsrequired for production of secondary metabolites. Penalva et al. (1998),Trends Biotechnol. 16: 483-489 discloses that production strains of P.chrysogenum have increased copy number of the penicillin synthesisstructural genes. Other studies have modulated expression of otherbiosynthetic enzyme-encoding genes, thereby affecting overall metabolismin the fungus. Mingo et al. (1999), J. Biol. Chem. 21: 14545-14550,demonstrate that disruption of phacA, an enzyme in A. nidulans thatcatalyzes phenylacetate 2-hydroxylation, leads to increased penicillinproduction, probably by elimination of competition for the substratephenylacetate. Similarly, disruption of the gene encoding aminoadipatereductase in P. chrysogenum increased penicillin production, presumablyby eliminating competition for the substrate alpha-aminoadipate(Casquiero et al. (1999), J. Bacteriol. 181: 1181-1188).

[0007] Thus, genetic manipulation holds promise for improving productionof secondary metabolites. Genetic manipulation to increase the activityof biosynthetic enzymes for secondary metabolite production or todecrease the activity of competing biosynthetic pathways has proveneffective for improving production. Maximum benefit can be achieved bycombining several strategies of manipulation. For example, modulatingthe expression of genes that regulate the biosynthetic enzyme-encodinggenes can improve production. In addition, genetic manipulation can beused to impact upon the challenges that are encountered in the fermentorrun or downstream processing (e.g., energy cost, specific production ofdesired metabolite, maximal recovery of metabolite, cost of processingwaste from fermentations). There is, therefore, a need for methods forimproving secondary metabolite production in a fungus, comprisingmodulating the expression of a gene involved in regulation of secondarymetabolite production in fungi.

[0008] One challenge is to identify the types of genes that would beuseful for such modulation. Todd and Andrianopoulos (1997), FungalGenetics and Biology 21: 388-405, teaches that Zn(II)2Cys6 proteins(zinc binuclear cluster proteins, or “ZBC proteins”) are involved in awide range of processes, including primary and secondary metabolism.This reference teaches that such proteins are primarily, though notexclusively, transcriptional activators. Chang et al. (1995), AppliedEnviron. Microbiol. 61: 2372-2377, teaches that increased expression ofaflR, a ZBC protein, relieves nitrate inhibition of aflatoxinbiosynthesis in Aspergillus parasiticus. PCT Publication WO 00/37629,teaches that over-expressing lovE, another ZBC protein, increaseslovastatin production in Aspergillus terreus. Noel et al (1998), Mol.Microbiol. 27: 131-142, teaches that xinR, a ZBC protein, inducesexpression of xylanolytic extracellular enzymes in Aspergillus niger.Hasper et al. (2000), Mol. Microbiol. 36: 193-200, teaches that xlnRalso regulates D-xylose reductase gene expression in Aspergillus niger.D'Alessio and Brandriss (2000), J. Bacteriology 182: 3748-3753,discloses that Gal4p, a ZBC protein, can activate the PUT (prolineutilization) genes in a Saccharomyces cerevisiae strain lacking thenormal gene for regulation of this pathway, PUT3. PCT Publication WO00/20596 discloses that prtT, a ZBC protein, activates extracellularproteases in Aspergillus niger.

[0009] Numerous studies have examined the effects of mutations in genesthat encode ZBC proteins. Crowley et al (1998), J. Bacteriol. 180:4177-4183, discloses that a single missense mutation in UPC2, a ZBCgene, results in pleiotropic effects in Saccharomyces cerevisiae. Fridenet al. (1989), Mol. Cell. Biol. 9: 4056-4060, teaches that a largeinternal deletion in Leu3p, a ZBC protein, in Sacclzaromyces cerevisiaecauses the protein to be a constitutive transcriptional activator.Oestreicher and Scazzocchio (1995), J. MoL Biol. 249: 693-699, disclosesthat a single amino acid change in Yc462, a ZBC protein, leads toconstitutive, hyperinducible and derepressed expression of at leastthree genes in Aspergillus nidulans. Wang et al. (1999), J. Biol. Chem.274: 19017-19024, discloses that nine distinct missense mutations inLEU3 affect the masking of the activation domain of that ZBC protein.Dickson et al. (1990), Nucleic Acids Res. 18: 5213-5217, discloses thatsingle amino acid changes in the C terminal region of Gal4p and Lac9p,two ZBC proteins, lead to constitutive expression of target genes.Marczak and Brandriss (1991), MoL CelL Biol. 11: 2609-2619, teaches thatsingle point mutations in PUT3, a ZBC gene from Saccharomycescerevisiae, lead to either constitutive or uninducible expression ofproline utilization genes. Carvajal et al. (1997), Mol. Gen. Genet. 256:406-415, teaches that single amino acid substitutions in Pdr1p, a ZBCprotein from Saccharomyces cerevisiae, are responsible forover-expression of three transporter genes associated with multiple drugresistance. Nourani et al. (1997), Mol. Gen. Genet. 256: 397-405,teaches that substitutions in a conserved region of Pdr3p, a ZBC proteinfrom Saccharomyces cerevisiae, leads to gain of function mutations. Zhouet al. (1990), Nucleic Acids Res. 18: 291-298, discloses that deletionof all or part of the linker region of Leu3p results in unmodulatedactivation of Leu3p target genes. Herlich et al. (1998), Fungal GeneticsBiol. 23: 1807-1845, teaches that deletion of three amino acids in the Cterminus of Af1R results in increased expression of the aflatoxinpathway.

[0010] These studies demonstrate that ZBC genes can be manipulated inbeneficial ways and may have promise as regulators of secondarymetabolism. Unfortunately, no one has been able to create a commercialprocess in which production of a useful secondary metabolite has beensignificantly increased through the action of a ZBC protein. There is,therefore, a need for new commercial processes using ZBC proteins, orvariants thereof, to significantly increase useful secondary metaboliteproduction.

SUMMARY OF THE INVENTION

[0011] The invention relates to secondary metabolite production byfungi. More particularly, the invention relates to modulation ofsecondary metabolite production by fungi through genetic manipulation ofsuch fungi. The invention provides new commercial processes using ZBCproteins, or variants thereof, to significantly improve the productionof useful secondary metabolites. Generally, the methods according to theinvention comprise expressing in a fungus a ZBC protein or a variantthereof.

[0012] In a first aspect, the invention provides methods for improvingproduction of a secondary metabolite by a fungus by increasing the yieldof the secondary metabolite produced by the fungus. The methodsaccording to this aspect of the invention comprise modulating theexpression of a ZBC gene or gene variant in a manner that improves theyield of the secondary metabolite.

[0013] In a second aspect, the invention provides methods for improvingproduction of a secondary metabolite by a fungus by increasingproductivity of the secondary metabolite in the fungus, the methodscomprising modulating the expression of a ZBC gene or gene variant in amanner that improves the productivity of the secondary metabolite.

[0014] In a third aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by increasing efflux orexcretion of the secondary metabolite, the method comprising modulatingthe expression of a ZBC gene or gene variant in a manner that increasesefflux or excretion of the secondary metabolite.

[0015] In a fourth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by decreasingproduction of side products or non-desired secondary metabolites, themethod comprising modulating the expression of a ZBC gene or genevariant in a manner that decreases production of side products ornon-desired secondary metabolites.

[0016] In a fifth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by altering thecharacteristics of the fungus in a manner that is beneficial to theproduction of the secondary metabolite, the method comprising modulatingthe expression of a ZBC gene or gene variant in a manner that alters thecharacteristics of the fungus.

[0017] In a sixth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by causing conditionallysis of the fungus, the method comprising modulating the expression ofa ZBC gene or gene variant in a manner that causes conditional lysis.

[0018] In a seventh aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by increasing theresistance of the fungus to the deleterious effects of exposure to asecondary metabolite made by the same organism, the method comprisingmodulating the expression of a ZBC gene or gene variant in a manner thatincreases resistance to the deleterious effects of exposure to asecondary metabolite.

[0019] In an eighth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by modulating theexpression of one or more genes, the method comprising modulating theexpression of a ZBC gene or gene variant that does not normally modulatethe expression of such gene or genes.

[0020] In a ninth aspect, the invention provides genetically modifiedfungi, wherein the genetically modified fungi have an ability to producesecondary metabolites and the ability of the genetically modified fungusto produce secondary metabolites has been improved by any of the methodsaccording to the invention.

[0021] In a tenth aspect, the invention provides a method for making asecondary metabolite, the method comprising culturing a geneticallymodified fungus according to the invention under conditions suitable forthe production of secondary metabolites.

DETAILED DESCRIPTION

[0022] The invention relates to secondary metabolite production byfungi. More particularly, the invention relates to modulation ofsecondary metabolite production by fungi through genetic manipulation ofsuch fungi. All issued patents, published and pending patentapplications, and other references cited herein reflect the level ofknowledge in this field and are hereby incorporated by reference intheir entirety. In case of any conflict between the teachings of a citedreference and this specification, the latter shall prevail.

[0023] The invention provides new commercial processes using ZBCproteins, or variants thereof, to significantly increase usefulsecondary metabolite production. Generally, the methods according to theinvention comprise expressing in a fungus a ZBC protein or a variantthereof. All aspects of the invention contemplate the modulation of oneor more ZBC genes in a fungal cell of interest.

[0024] In a first aspect, the invention provides methods for improvingproduction of a secondary metabolite by a fungus by increasing the yieldof the secondary metabolite produced by the fungus. The methodsaccording to this aspect of the invention comprise modulating theexpression of a ZBC gene or gene variant in a manner that improves theyield of the secondary metabolite.

[0025] As used for all aspects of the invention, the term “improvingproduction of a secondary metabolite” means positively impacting one ormore of the variables that affect the process of production of secondarymetabolites in a fungal fermentation. These variables include, withoutlimitation, the amount of secondary metabolite being produced, thevolume required for production of sufficient quantities, the cost of rawmaterials and energy, the time of fermentor run, the amount of wastethat must be processed after a fermentor run, the specific production ofthe desired metabolite, the percent of produced secondary metabolitethat can be recovered from the fermentation broth, and the resistance ofan organism producing a secondary metabolite to possible deleteriouseffects of contact with the secondary metabolite. Also for all aspects,the term “secondary metabolite” means a compound, derived from primarymetabolites, that is produced by an organism, is not a primarymetabolite, is not ethanol or a fusel alcohol, and is not required forgrowth under standard conditions. Secondary metabolites are derived fromintermediates of many pathways of primary metabolism. These pathwaysinclude, without limitation, pathways for biosynthesis of amino acids,the shikimic acid pathway for biosynthesis of aromatic amino acids, thepolyketide biosynthetic pathway from acetyl coenzyme A (CoA), themevalonic acid pathway from acetyl CoA, and pathways for biosynthesis ofpolysaccharides and peptidopolysaccharides. Secondary metabolisminvolves all primary pathways of carbon metabolism (Fungal Physiology,Chapter 9, pp 246-274, Griffm (ed.), John Wiley & Sons, Inc., New York,(1994)). “Secondary metabolites” also include intermediate compounds inthe biosynthetic pathway for a secondary metabolite that are dedicatedto the pathway for synthesis of the secondary metabolite. “Dedicated tothe pathway for synthesis of the secondary metabolite” means that oncethe intermediate is synthesized by the cell, the cell will not convertthe intermediate to a primary metabolite. “Intermediate compounds” alsoinclude secondary metabolite intermediate compounds which can beconverted to useful compounds by subsequent chemical conversion orsubsequent biotransformation. Nevertheless, providing improvedavailability of such intermediate compounds still leads to improvedproduction of the ultimate useful compound, which itself may be referredto herein as a secondary metabolite. The yeast Saccharomyces cerevisiaeis not known to produce secondary metabolites. The term “primarymetabolite” means a natural product that has an obvious role in thefunctioning of the relevant organism. Primary metabolites include,without limitation, compounds involved in the biosynthesis of lipids,carbohydrates, proteins, and nucleic acids. The term “increasing theyield of the secondary metabolite” means increasing the quantity of thesecondary metabolite present in the fermentation broth per unit volumeof fermentation broth.

[0026] The term “ZBC gene” means any gene encoding a protein having aspart of its structureCys-(Xaa)₂-Cys-(Xaa)₆-Cys-(Xaa)₅-₁₆-Cys-(Xaa)₂-Cys-(Xaa)₆-₈-Cys (seee.g., Todd and Andrianopoulos (1997), Fungal Genetics and Biology 21:388-405), wherein Xaa is any amino acid, each of which can be the sameor different. Preferred ZBC genes according to this aspect of theinvention include, without limitation, those genes identified in Table1, below, and any fungal homologs thereof. TABLE 1 Examples of preferredZBC genes SEQ Length Size ID Name (aa) (bp) Organism NO AAB05250_Hh 4551365 Fusarium solani 13 AAC98670_Ca 517 1551 Candida albicans 15 AC15_Nc865 2595 Neurospora crassa 17 acr2_Nc 595 1785 Neurospora crassa 19AF168613_4_Ap 491 1473 Aspergillus parasiticus 21 AF230811_1_Pg 974 2922Pyricularia grisea 23 aflR_Af 437 1311 Aspergillus flavus 25 AFLR_An 4331299 Aspergillus nidulans 27 aflR_Ao 384 1152 Aspergillus oryzae 29aflR_Ap 444 1332 Aspergillus parasiticus 31 alcR_An 821 2463 Aspergillusnidulans 33 AmdR-An 765 2295 Aspergillus nidulans 35 AmdR-Ao 735 2205Aspergillus oryzae 37 AmyR-Anig 579 1737 Aspergillus niger 39 amyRAn 6621986 Aspergillus nidulans 41 amyRAo 604 1812 Aspergillus oryzae 43An13_An 311 933 Aspergillus nidulans 45 ARG81_YEAST 880 2640Saccharomyces 47 cerevisiae ARGRII_YEAST 879 2637 Saccharomyces 49cerevisiae At18_At 397 1191 Aspergillus terreus 51 BAA21449_Sp 738 2214Schizosaccharomyces 53 pombe BAA87112_Sp 71 212 Schizosaccharomyces 55pombe BAA87304_Sp 188 564 Schizosaccharomyces 57 pombe C23783_Pa 4121236 Pichia anomala 59 CAA11231_Pa 529 1587 Pichia anomala 61CAA18305_Sp 867 2601 Schizosaccharomyces 63 pombe CAA18884_Sp 397 1191Schizosaccharomyces 65 pombe CAA19035_Sp 827 2481 Schizosaccharomyces 67pombe CAA19036_Sp 560 1680 Schizosaccharomyces 69 pombe CAA19070_Sp 5251575 Schizosaccharomyces 71 pombe CAA19171_Sp 743 2229Schizosaccharomyces 73 pombe CAA 19174_Sp 815 2445 Schizosaccharomyces75 pombe CAA20477_Sp 547 1641 Schizosaccharomyces 77 pombe CAA21815_Sp857 2571 Schizosaccharomyces 79 pombe CAA21917_Sp 594 1782Schizosaccharomyces 81 pombe CAA21921_Sp 595 1785 Schizosaccharomyces 83pombe CAA21933_Ca 510 1530 Candida albicans 85 CAA22445_Sp 480 1440Schizosaccharomyces 87 pombe CAA22655_Sp 767 2301 Schizosaccharomyces 89pombe CAA22853_Sp 736 2208 Schizosaccharomyces 91 pombe CAA92308_Sp 6031809 Schizosaccharomyces 93 pombe CAB16735_Sp 783 2349Schizosaccharomyces 95 pombe CAB52588_Sm 689 2067 Sordaria macrospora 97CAB59617_Sp 625 1875 Schizosaccharomyces 99 pombe CAB61777_Sp 654 1962Schizosaccharomyces 101 pombe CAB71797_(—) 905 2715 Tolypocladiuminflatum 103 Tolypoclad CAR80_YEAST 836 2508 Saccharomyces 105cerevisiae CAT8_Ca 1056 3168 Candida albicans 107 CAT8_Kl 1445 4335Kluyveromyces lactis 109 CAT8_YEAST 1433 4299 Saccharomyces 111cerevisiae CEP3_YEAST 608 1824 Saccharomyces 113 cerevisiae CHA4_YEAST648 1944 Saccharomyces 115 cerevisiae CMR1_Cl 984 2952 Colletotrichum117 lagenarium CT1A_Fs 909 2727 Haematonectria 119 haematococca CT1B_Fs882 2646 Haematonectria 121 haematococca CZF1_Ca 388 1164 Candidaalbicans 123 DAL81_YEAST 970 2910 Saccharomyces 125 cerevisiaeECM22_YEAST 814 2442 Saccharomyces 127 cerevisiae FacB_An 867 2601Aspergillus nidulans 129 FacB_Anig 862 2586 Aspergillus niger 131FacB_Ao 859 2577 Aspergillus oryzae 133 FLUF_Nc 792 2376 Neurosporacrassa 135 GAL4_YEAST 881 2643 Saccharomyces 137 cerevisiae HAL9_YEAST1030 3090 Saccharomyces 139 cerevisiae HAP1_1483YEAST 1483 4449Saccharomyces 141 cerevisiae HAP1_1502YEAST 1502 4506 Saccharomyces 143cerevisiae LAC9_Kl 865 2595 Kluyveromyces lactis 145 lac9_Kmarx 865 2595Kluyveromyces 147 marxianus var. lactis LEU3_YEAST 886 2658Saccharomyces 149 cerevisiae lovE_At 503 1509 Aspergillus terreus 151lovEv2_At 469 1492 Aspergillus terreus 153 lovU_At 742 2226 Aspergillusterreus 155 LYS14_YEAST 790 2370 Saccharomyces 157 cerevisiaeM81157_1_Spast 470 1410 Saccharomyces 159 or pastorianus MAL13_YEAST 4731419 Saccharomyces 161 cerevisiae MAL23_YEAST 470 1410 Saccharomyces 163cerevisiae MAL33_YEAST 468 1404 Saccharomyces 165 cerevisiae MAL63_YEAST470 1410 Saccharomyces 167 cerevisiae MAL6_Scarlsberg 473 1419Saccharomyces 169 carlsbergensis MSP8_YEAST 1429 4287 Saccharomyces 171cerevisiae NIRA_An 892 2676 Aspergillus nidulans 173 NIT4_Nc 1090 3270Neurospora crassa 175 ntf1/thi1_Sp 775 2325 Schizosaccharomyces 177pombe OAF1_YEAST 1062 3186 Saccharomyces 179 cerevisiae PDR3_YEAST 9762928 Saccharomyces 181 cerevisiae PIP2_YEAST 996 2988 Saccharomyces 183cerevisiae PPR1_YEAST 904 2712 Saccharomyces 185 cerevisiae PRIB_Le 5651695 Shiitake mushroom 187 prnA_An 818 2454 Aspergillus nidulans 189PUT3_YEAST 979 2937 Saccharomyces 191 cerevisiae QA1F_Nc 816 2448Neurospora crassa 193 QUTA_An 825 2475 Aspergillus nidulans 195RGT1_YEAST 1170 3510 Saccharomyces 197 cerevisiae SEF1_K1 1071 3213Kluyveromyces lactis 199 SEF1_YEAST 1057 3171 Saccharomyces 201cerevisiae SIP4_YEAST 829 2487 Saccharomyces 203 cerevisiae STB4_YEAST949 2847 Saccharomyces 205 cerevisiae STB5_YEAST 743 2229 Saccharomyces207 cerevisiae SUC1_Ca 501 1503 Candida albicans 209 SUT1_YEAST 299 897Saccharomyces 211 cerevisiae TamA_An 739 2217 Aspergillus nidulans 213TBS1_YEAST 1094 3282 Saccharomyces 215 cerevisiae TEA1_YEAST 759 2277Saccharomyces 217 cerevisiae THI2_YEAST 450 1350 Saccharomyces 219cerevisiae UAY_An 1060 3180 Aspergillus nidulans 221 UGA3_YEAST 528 1584Saccharomyces 223 cerevisiae xlnR_Anig 875 2625 Aspergillus niger 225YAKB_Sp 782 2346 Schizosaccharomyces 227 pombe YAO7_Sp 637 1911Schizosaccharomyces 229 pombe YAOC_Sp 357 1071 Schizosaccharomyces 231pombe YAOClong_Sp 644 1932 Schizosaccharomyces 233 pombe YAS8_Sp 5631689 Schizosaccharomyces 235 pombe YBR033W_YEAST 919 2757 Saccharomyces237 cerevisiae YBR239C_YEAST 529 1587 Saccharomyces 239 cerevisiaeYCR106W_YEAST 832 2496 Saccharomyces 241 cerevisiae YDR213W_YEAST 9132739 Saccharomyces 243 cerevisiae YDR303C_YEAST 885 2655 Saccharomyces245 cerevisiae YDR421W_YEAST 950 2850 Saccharomyces 247 cerevisiaeYDR520C_YEAST 772 2316 Saccharomyces 249 cerevisiae YER184C_YEAST 7942382 Saccharomyces 251 cerevisiae YFL052W_YEAST 465 1395 Saccharomyces253 cerevisiae YIL130W_YEAST 964 2892 Saccharomyces 255 cerevisiaeYJL103C_YEAST 618 1854 Saccharomyces 257 cerevisiae YJL206C_YEAST 7582274 Saccharomyces 259 cerevisiae YKL222C_YEAST 705 2115 Saccharomyces261 cerevisiae YKR064W_YEAST 863 2589 Saccharomyces 263 cerevisiaeYLL054C_YEAST 769 2307 Saccharomyces 265 cerevisiae YLR266C_YEAST 7012103 Saccharomyces 267 cerevisiae YLR278C_YEAST 1341 4023 Saccharomyces269 cerevisiae YML076C_YEAST 944 2832 Saccharomyces 271 cerevisiaeYNR063W_YEAST 607 1821 Saccharomyces 273 cerevisiae YOR172W_YEAST 7862358 Saccharomyces 275 cerevisiae YOR380W_YEAST 546 1638 Saccharomyces277 cerevisiae YPL133C_YEAST 446 1338 Saccharomyces 279 cerevisiaeYPR009W_YEAST 268 804 Saccharomyces 281 cerevisiae YPR196W_YEAST 4701410 Saccharomyces 283 cerevisiae YRR1_YEAST 810 2430 Saccharomyces 285cerevisiae ZNF1_Ca 388 1164 Candida albicans 287 CAB57441_Sp 497 1491Schizosaccharomyces 289 pombe PDR1SGD_YEAST 1068 3204 Saccharomyces 291cerevisiae PDR1_YEAST 1063 3189 Saccharomyces 293 cerevisiae YHL6_YEAST883 2649 Saccharomyces 295 cerevisiae At233 309 927 Aspergillus terreus297 Pc1001 859 2577 Penicillium 299 chrysogenum At274 424 1272Aspergillus terreus 301 At221 850 2550 Aspergillus terreus 303 An1000758 2274 Aspergillus nidulans 305 At240 576 1728 Aspergillus terreus 307

[0027] A “fungal homolog” of a reference gene is a fungal gene encodinga product that is capable of performing at least a portion of thefunction of the product encoded by the reference gene, which issubstantially identical to the reference gene, and/or which encodes aproduct which is substantially identical to the product encoded by thereference gene. “Substantially identical” means a polypeptide or nucleicacid exhibiting at least 25%, preferably 50%, more preferably 80%, andmost preferably 90%, or even 95% identity to a reference amino acidsequence or nucleic acid sequence. For polypeptides, the length ofcomparison sequences is generally at least 16 amino acids, preferably atleast 20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids or greater. For nucleic acids, the length ofcomparison sequences is generally at least 50 nucleotides, preferably atleast 60 nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides or greater. Sequence identity is typicallymeasured using sequence analysis software (for example, the SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison Wis.53705; or the BLAST, BEAUTY, or PILEUP/PRETTYBOX programs). Fordetermining percentages of identity, a gap existence penalty of 11 and agap extension penalty of 1 may be employed in such programs. Fordetermining sequence similarity, such software assigns degrees ofhomology to various substitutions, deletions, and/or othermodifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine, valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

[0028] For this aspect of the invention, when the secondary metaboliteis aflatoxin or sterigmatocystin, the ZBC gene is not the aflR gene fromAspergillus spp.; and when the secondary metabolite is lovastatin, theZBC gene is not the Aspergillus terreus lovE gene.

[0029] The term “ZBC gene or gene variant” means any ZBC gene, or anyuseful mutant form of such gene. Many useful mutations of ZBC genesand/or proteins are contemplated by the present invention, includingvarious dominant mutations. A “dominant mutation” is an allele of a genethat encodes a protein capable of changing the phenotype of an organismmore than a non-mutated form of the gene. Preferred dominant mutationsinclude dominant negative mutations, dominant positive mutations, anddominant neomorphic mutations. A “dominant negative mutation” is adominant mutation that achieves its phenotypic effect by interferingwith some function of the gene or gene product from which it wasderived, or from a homolog thereof. A “dominant positive mutation” is adominant mutation that achieves its phenotypic effect by activating somefunction of the gene or gene product from which it was derived, or froma homolog thereof. A “dominant neomorphic mutation” is a dominantmutation that achieves the phenotypic effect of providing a novelfunction to the gene or gene product from which it was derived, or froma homolog thereof. Preferred dominant mutations according to this aspectof the invention include:

[0030] (1) Mutations that result in increased or decreased stability ofthe transcript of a gene.

[0031] (2) Mutations that result in increased or decreased stability ofthe product of translation: For example, specific sequences near theamino terminus of a protein have been shown to cause increased ordecreased protein stability. Similarly, sequences elsewhere in theprotein, such as those required for ubiquitin-dependent degradation, canbe mutated to increase the stability of a protein.

[0032] (3) Amino acid substitutions that mimic post-translationalmodifications: For example, phosphorylation has been demonstrated topositively or negatively regulate the activity of a variety of proteins,including transcription factors and kinases. Phosphorylation mostcommonly occurs on serine, threonine, and tyrosine residues; in someinstances residues such as aspartate and histidine can bephosphorylated. Mutations that mimic constitutive dephosphorylation canbe produced by mutating the coding sequence of the phosphorylatedresidue to the coding sequence of an amino acid that cannot bephosphorylated and does not have a negatively charged side chain (e.g.,alanine). Alternatively, substitutions that result in changing serine,threonine, or tyrosine residues to charged amino acids such as glutamateor aspartate can result in an allele that mimics constitutivephosphorylation.

[0033] Proteolytic cleavage is another post-translational mechanism forregulating the activity of a protein. Mutations that result intruncation of a protein can mimic activation by proteolysis. Mutationsthat change amino acids required for proteolysis can activate proteinsthat are negatively regulated by proteolysis.

[0034] (4) Amino acid substitutions that promote or inhibit the bindingof small molecules such as ATP, cAMP, GTP or GDP: For example,Nucleotides are co-factors for many enzymes, and the nucleotide-bindingdomains of such proteins are highly conserved. Lysine to argininesubstitutions in the nucleotide binding domain frequently result in theinhibition of enzymatic activity. Enzymatically inactive proteins can bedominant inactive molecules, acting by sequestering substrates fromfunctional enzymes.

[0035] (5) Mutations in portions of genes that encode regulatory domainsof proteins: For example, many proteins, including kinases, containregulatory domains that function as mechanisms to control the timing ofactivation.

[0036] Mutations in these domains might result in constitutiveactivation. Regulatory domains include linker regions and C terminalregions in the case of some ZBC proteins.

[0037] (6) Mutations that create a new protein function: For example, amutation in a ZBC protein could result in altered DNA recognitionspecificity, such that the mutated ZBC protein can modulate the activityof pathways that it does not usually regulate.

[0038] (7) Fusion of the ZBC protein or variants thereof to atranscriptional activation domain: Transcriptional activation domains(TADS) are defined as discrete regions of proteins that promote geneexpression by a variety of mechanisms that ultimately result in theactivation of RNA polymerase. A TAD generally is defined as the minimalmotif that activates transcription when fused to a DNA-binding domain(Webster et al. (1988), Cell 52: 169-178; Fischer et al. (1988), Nature332: 853-856; Hope et al. (1988), Nature 333: 635-640).

[0039] As used for all aspects of the invention, the term “modulatingthe expression of a gene” means affecting the function of a gene'sproduct, preferably by increasing or decreasing protein activity orcreating a new protein activity through mutation; increasing ordecreasing transcription; increasing or decreasing translation;increasing, decreasing or changing post-translational modification;altering intracellular localization; increasing or decreasingtranslocation; increasing or decreasing protein activity by fusion or byinteraction of the protein with another molecule; and/or creating a newprotein activity by interaction of the protein with another molecule. Insome cases, such modulation is achieved by allowing or causing theexpression of an exogenously supplied nucleic acid or gene, e.g., bytransformation. In some cases, other exogenously supplied molecules canmediate the modulation. The modulation is not achieved, however, bysimply randomly mutagenizing the fungus, either spontaneously or bychemical means. In certain embodiments, the ZBC gene is from an organismin which it is not present within a biosynthetic cluster, or the ZBCgene is not present in the biosynthetic cluster of the desired secondarymetabolite to be regulated. In certain embodiments, the ZBC gene is froman organism other than the production fungus, preferably from adifferent species or genus. In certain embodiments, the ZBC gene in itsnative locus regulates a different secondary metabolite than the desiredsecondary metabolite produced by the production fungus. In certainembodiments, the ZBC gene in its native locus does not regulatesecondary metabolism. “Native locus” means the chromosomal locus in theoriginal organism from which the gene was cloned.

[0040] As used for all aspects of the invention, “mutation” means analteration in DNA sequence, either by site-directed or randommutagenesis. Mutation encompasses point mutations as well as insertions,deletions, or rearrangements.

[0041] As used for all aspects of the invention, “mutant” means anorganism containing one or more mutations.

[0042] In certain embodiments of the methods according to this aspect ofthe invention, the modulation is over-expression of the gene.“Over-expression of the gene” means transcription and/or translationand/or gene product maturation at a rate that exceeds by at leasttwo-fold, preferably at least five-fold, and more preferably at leastten-fold, the level of such expression that would be present undersimilar growth conditions in the absence of the modulation of expressionof the gene. “Similar growth conditions” means similar sources ofnutrients such as carbon, nitrogen, and phosphate, as well as similarpH, partial oxygen pressure, temperature, concentration of drugs orother small molecules, and a similar substrate for growth, whethersolid, semi-solid, or liquid.

[0043] In certain embodiments of the methods according to this aspect ofthe invention, the modulation is expression of a dominant mutation ofthe gene. The term “dominant mutation” is as used before. Preferreddominant mutations according to this aspect of the invention are as usedbefore.

[0044] In certain embodiments of the methods according to this aspect ofthe invention, the modulation is conditional expression of the gene.“Conditional expression” of a gene means expression under certain growthconditions, but not under others. Such growth conditions that may bevaried include, without limitation, carbon source, nitrogen source,phosphate source, pH, temperature, partial oxygen pressure, the presenceor absence of small molecules such as drugs, and the presence or absenceof a solid substrate.

[0045] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an anti-bacterial. An“anti-bacterial” is a molecule that has cytocidal or cytostatic activityagainst some or all bacteria. Preferred anti-bacterials include, withoutlimitation, β-lactams. Preferred β-lactams include, without limitation,penicillins and cephalosporins. Preferred penicillins and biosyntheticintermediates include, without limitation, isopenicillin N,6-aminopenicillanic acid (6-APA), penicillin G, penicillin N, andpenicillin V. Preferred cephalosporins and biosynthetic intermediatesinclude, without limitation, deacetoxycephalosporin V (DAOC V),deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC),7-aminodeacetoxy-cephalosporanic acid (7-ADCA), cephalosporin C,7-β-(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7ACA),7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA), and7-aminocephalosporanic acid (7ACA).

[0046] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an anti-hypercholesterolemic.An “anti-hypercholesterolemic” is a drug administered to a patientdiagnosed with elevated cholesterol levels, for the purpose of loweringthe cholesterol levels. Preferred anti-hypercholesterolemics include,without limitation, lovastatin, mevastatin, simvastatin, andpravastatin.

[0047] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an immunosuppressant. An“immunosuppressant” is a molecule that reduces or eliminates an immuneresponse in a host when the host is challenged with an immunogenicmolecule, including immunogenic molecules present on transplantedorgans, tissues or cells. Preferred immunosuppressants include, withoutlimitation, members of the cyclosporin family and beauverolide L.Preferred cyclosporins include, without limitation, cyclosporin A andcyclosporin C.

[0048] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an ergot alkaloid. An “ergotalkaloid” is a member of a large family of alkaloid compounds that aremost often produced in the sclerotia of fungi of the genus Claviceps. An“alkaloid” is a small molecule that contains nitrogen and has basic pHcharacteristics. The classes of ergot alkaloids include clavinealkaloids, lysergic acids, lysergic acid amides, and ergot peptidealkaloids. Preferred ergot alkaloids include, without limitation,ergotamine, ergosine, ergocristine, ergocryptine, ergocornine,ergotaminine, ergosinine, ergocristinine, ergocryptinine, ergocorninine,ergonovine, ergometrinine, and ergoclavine.

[0049] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an inhibitor of angiogenesis.An “angiogenesis inhibitor” is a molecule that decreases or prevents theformation of new blood vessels. Angiogenesis inhibitors have proveneffective in the treatment of several human diseases including, withoutlimitation, cancer, rheumatoid arthritis, and diabetic retinopathy.Preferred inhibitors of angiogenesis include, without limitation,fumagillin and ovalicin.

[0050] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a glucan synthase inhibitor.A “glucan synthase inhibitor” is a molecule that decreases or inhibitsthe production of 1,3-β-D-glucan, a structural polymer of fungal cellwalls. Glucan synthase inhibitors are a class of antifungal agents.Preferred glucan synthase inhibitors include, without limitation,echinocandin B, pneumocandin B, aculeacin A, and papulacandin.

[0051] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a member of the gliotoxinfamily of compounds. The “gliotoxin family of compounds” are relatedmolecules of the epipolythiodioxopiperazine class. Gliotoxins displaydiverse biological activities, including, without limitation,antimicrobial, antifungal, antiviral, and immunomodulating activities.Preferred members of the “gliotoxin family of compounds” include,without limitation, gliotoxin and aspirochlorine.

[0052] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a fungal toxin. A “fungaltoxin” is a compound that causes a pathological condition in a host,either plant or animal. Fungal toxins could be mycotoxins present infood products, toxins produced by phytopathogens, toxins from poisonousmushrooms, or toxins produced by zoopathogens. Preferred fungal toxinsinclude, without limitation, aflatoxins, patulin, zearalenone,cytochalasin, griseofulvin, ergochrome, cercosporin, marticin,xanthocillin, coumarins, tricothecenes, fusidanes, sesterpenes,amatoxins, malformin A, phallotoxins, pentoxin, HC toxin, psilocybin,bufotenine, lysergic acid, sporodesmin, pulcheriminic acid, sordarins,fumonisins, ochratoxin A, and fusaric acid.

[0053] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a modulator of cell surfacereceptor signaling. As used herein, the term “cell surface receptor”means a molecule that resides at or in the plasma membrane, binds anextracellular signaling molecule, and transduces this signal topropagate a cellular response. Modulators of cell surface receptorsignaling might function by one of several mechanisms including, withoutlimitation, acting as agonists or antagonists; sequestering a moleculethat interacts with a receptor, such as a ligand; or stabilizing theinteraction of a receptor and a molecule with which it interacts.Preferred modulators of cell surface signaling include, withoutlimitation, the insulin receptor agonist L-783,281 and thecholecystokinin receptor antagonist asperlicin.

[0054] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a plant growth regulator. A“plant growth regulator” is a molecule that controls growth anddevelopment of a plant by affecting processes that include, withoutlimitation, division, elongation, and differentiation of cells.Preferred plant growth regulators include, without limitation,cytokinin, auxin, gibberellin, abscisic acid, and ethylene.

[0055] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is a pigment. A “pigment” is asubstance that imparts a characteristic color. Preferred pigmentsinclude, without limitation, melanins and carotenoids.

[0056] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an insecticide. An“insecticide” is a molecule that is toxic to at least some insects. Apreferred insecticide, without limitation, is nodulisporic acid.

[0057] In certain embodiments of the methods according to this aspect ofthe invention, the secondary metabolite is an anti-neoplastic compound.An “anti-neoplastic” compound is a molecule that prevents or reducestumor formation. Preferred anti-neoplastic compounds include, withoutlimitation, taxol (paclitaxel) and related taxoids.

[0058] In certain embodiments of the methods according to this aspect ofthe invention, the methods further comprise purifying the secondarymetabolite from a culture of the fungus. “Purifying” means obtaining thesecondary metabolite in substantially pure form. “Substantially pure”means comprising at least 90%, more preferably at least 95%, and mostpreferably at least 99%, of the purified composition on a dry-weightbasis.

[0059] In a second aspect, the invention provides methods for improvingproduction of a secondary metabolite by a fungus by increasingproductivity of the secondary metabolite in the fungus, the methodscomprising modulating the expression of a ZBC gene or gene variant in amanner that improves the productivity of the secondary metabolite.

[0060] “Improves the productivity” means to increase the quotient of theconcentration of the secondary metabolite divided by the product of thefermentor run-time multiplied by the fermentation volume multiplied bythe grams of the dry cell weight of biomass (Productivity=concentrationmetabolite/(time×volume ×gDCW)).

[0061] Significant advantages that might result from increasingproductivity include, without limitation, a decrease in fermentorrun-time, a decrease in the size of the fermentor required forproduction of equivalent amounts of secondary metabolite, or a decreasein the biomass required for production, which translates into decreasedwaste that must be handled in downstream processing. Preferably, suchincreased productivity is by at least ten percent, more preferably atleast 50 percent, and most preferably at least two-fold.

[0062] “Modulating the expression of a ZBC gene” is as used before. Incertain embodiments of the methods according to this aspect of theinvention, the modulation is over-expression of the ZBC gene.“Over-expression of the gene” is as used before. In certain embodimentsof the methods according to this aspect of the invention, the modulationis expression of a dominant mutation of the gene. The term “dominantmutation” is as used before. Preferred dominant mutations according tothis aspect of the invention are as used before. In certain embodimentsof the methods according to this aspect of the invention, the modulationis conditional expression of the gene. The term “conditional expression”of a gene is as used before.

[0063] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0064] In a third aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by increasing efflux orexcretion of the secondary metabolite, the method comprising modulatingthe expression of a ZBC gene or gene variant in a manner that increasesefflux or excretion of the secondary metabolite. “Increasing efflux orexcretion of the secondary metabolite” means that, without lysing afungal cell, a greater quantity of the secondary metabolite passes fromthe inside of the fungal cell to the outside of the fungal cell per unittime. “Outside of the fungal cell” is defined as being no longercontained wholly within the lipid bilayer of the cell and/or extractablefrom the cell with methods which do not release a majority ofintracellular contents. “Modulating the expression of a ZBC gene” is asused before, except that the ZBC gene can be the Aspergillus spp. aflRgene when the secondary metabolite is aflatoxin or sterigmatocystin, andthe ZBC gene can be lovE when the secondary metabolite is lovastatin. Incertain embodiments of the methods according to this aspect of theinvention, the modulation is over-expression of the gene.“Over-expression of the gene” is as used before. In certain embodimentsof the methods according to this aspect of the invention, the modulationis expression of a dominant mutation of the gene. The term “dominantmutation” is as used before. Preferred dominant mutations according tothis aspect of the invention are as used before. In certain-embodimentsof the methods according to this aspect of the invention, the modulationis conditional expression of the gene. The term “conditional expression”of a gene is as used before.

[0065] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0066] In a fourth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by decreasingproduction of side products or non-desired secondary metabolites, themethod comprising modulating the expression of a ZBC gene or genevariant in a manner that decreases production of side products ornon-desired secondary metabolites. “Decreasing production of sideproducts or non-desired secondary metabolites” means reducing the amountof such side products or non-desired secondary metabolites that aresynthesized or which are retained within the cells or the mediasurrounding the cells. Preferably, such reduction is at least by 25%,more preferably by at least 50%, even more preferably by at least2-fold, and most preferably by at least 5-fold. “Modulating theexpression of a ZBC gene” is as used for the third aspect of theinvention. In certain embodiments of the methods according to thisaspect of the invention, the modulation is over-expression of the gene.“Over-expression of the gene” is as used before. In certain embodimentsof the methods according to this aspect of the invention, the modulationis expression of a dominant mutation of the gene. The term “dominantmutation” is as used before. Preferred dominant mutations according tothis aspect of the invention are as used before. In certain embodimentsof the methods according to this aspect of the invention, the modulationis conditional expression of the gene. The term “conditional expression”of a gene is as used before.

[0067] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0068] In a fifth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by altering thecharacteristics of the fungus in a manner that is beneficial to theproduction of the secondary metabolite, the method comprising modulatingthe expression of a ZBC gene or gene variant in a manner that alters thecharacteristics of the fungus.

[0069] “Altering the characteristics” means changing the morphology orgrowth traits of the fungus. Preferred alterations include, withoutlimitation, alterations that result in transition of the fungus from thehyphal to the yeast form; alterations that result in transition of thefungus from the yeast to the hyphal form; alterations that lead to moreor less hyphal branching; alterations that increase or decreaseflocculence, adherence, cell buoyancy, surface area of the fungus, cellwall integrity and/or stability, pellet size, vacuole formation, and/orability to grow at higher or lower temperatures; and alterations thatincrease the saturating growth density of a culture or rate of pelletformation. “Modulating the expression of a ZBC gene” is as used for thethird aspect of the invention. In certain embodiments of the methodsaccording to this aspect of the invention, the modulation isover-expression of the gene. “Over-expression of the gene” is as usedbefore. In certain embodiments of the methods according to this aspectof the invention, the modulation is expression of a dominant mutation ofthe gene. The term “dominant mutation” is as used before. Preferreddominant mutations according to this aspect of the invention are as usedbefore. In certain embodiments of the methods according to this aspectof the invention, the modulation is conditional expression of the gene.The term “conditional expression” of a gene is as used before.

[0070] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0071] In a sixth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by causing conditionallysis of the fungus, the method comprising modulating the expression ofa ZBC gene or gene variant in a manner that causes conditional lysis.“Causing conditional lysis” means causing the fungus to grow withoutlysis under a first set of growth conditions and to lyse under a secondand different set of conditions, which are not lytic to the unmodifiedfungus. In preferred embodiments, the conditions that can be alteredbetween the first and second growth conditions include, withoutlimitation, the source or amount of nutrients such as carbon, nitrogen,and phosphate; the source or amount of specific enzymes; the source oramount of specific components found in cell walls; the amount of saltsor osmolytes; the pH of the medium; the partial oxygen pressure;temperature; and the amount of specific small molecules.

[0072] “Modulating the expression of a ZBC gene” is as used for thethird aspect of the invention. In certain embodiments of the methodsaccording to this aspect of the invention, the modulation isover-expression of the gene. “Over-expression of the gene” is as usedbefore. In certain embodiments of the methods according to this aspectof the invention, the modulation is expression of a dominant mutation ofthe gene. The term “dominant mutation” is as used before. Preferreddominant mutations according to this aspect of the invention are as usedbefore. In certain embodiments of the methods according to this aspectof the invention, the modulation is conditional expression of the gene.The term “conditional expression” of a gene is as used before.

[0073] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0074] In a seventh aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by increasing theresistance of the fungus to the deleterious effects of-exposure to asecondary metabolite made by-the same organism, the method comprisingmodulating the expression of a ZBC gene or gene variant in a manner thatincreases resistance to the deleterious effects of exposure to asecondary metabolite. As used herein, the phrase “increasing theresistance of the fungus to the deleterious effects of exposure to asecondary metabolite” means to allow the fungus to survive, grow, orproduce the secondary metabolite in conditions that otherwise would betoxic to the fungus or prevent the production of the secondarymetabolite. In particular, the growth of a fungus that produces asecondary metabolite can be limited, in part, by the toxic effects ofthe secondary metabolite itself. In the absence of resistance mechanismsto protect the fungi from the toxic effects of these metabolites,decreased yields of the metabolite can be observed. For example,Alexander et al. (1999), Mol. Gen. Genet. 261: 977-84, have shown thatthe trichothecene efflux pump of Fusarium sporotrichiodes, encoded bythe gene TR112, is required both for high level production of, andresistance to the toxic effects of, trichothecenes produced by thisfungus. Thus, modifications that increase the resistance of a fungus toa toxic secondary metabolite that it produces can increase thesaturation density and extend the metabolically active lifetime of theproducing fungus. In a bioreactor, such attributes will have thebeneficial effect of increasing the yield and productivity of ametabolite. Regulators of secondary metabolite production whoseexpression can be modulated to increase resistance of a fungus to toxicmetabolites can include, without limitation, transporters that promoteefflux of the metabolite from cells, enzymes that alter the chemicalstructure of the metabolite within cells to render it non-toxic,target(s) of the metabolite that mediate its toxicity, and gene productsthat alter cellular processes to counteract the toxic effects of ametabolite. Additional benefits of increasing efflux of secondarymetabolites include increasing the amount of metabolite available forpurification from the fermentation broth and mitigation of feedbackinhibition of secondary metabolism that may be mediated by themetabolite itself. Indeed, feedback inhibition of a biosynthetic pathwayby a product of that pathway is well documented in many microorganisms,and this inhibition can act at the transcriptional, translational, andpost-translational levels. Several well-documented examples in yeastinclude the transcriptional repression of lysine biosynthetic genes bylysine (Feller et al. (1999), Eur. J. Biochen. 261: 163-70), thedecreased stability of both the mRNA encoding the uracil permease Fur4pand the permease itself in the presence of uracil (Seron et al. (1999),J. Bacteriol. 181: 1793-800), and the inhibition of alpha-isopropylmalate synthase, a key step in leucine biosynthesis, by the presence ofleucine (Beltzer et aL (1988), J. Biol. Chem. 263:368-74).

[0075] Transcription factors that regulate the expression of effluxpumps could also be used to increase efflux of a drug from a fungal cellto increase the yields of a metabolite and decrease the toxicity of thesecondary metabolite in a fermentation. Such transcription factorsinclude, but are not limited to, ZBC genes such as PDR1, and PDR3 fromS. cerevisiae and their homologs. Over-expression of each of these geneshas been shown to up-regulate expression of transporters and causeincreased resistance of S. cerevisiae to toxic compounds (for examples,see Reid et al. (1997), J. Biol. Chem. 272: 12091-9; Katzmann et al.(1994), Mol. Cell. Biol. 14: 4653-61; Wendler et al. (1997), J. Biol.Chem. 272: 27091-8).

[0076] Increases in resistance to the toxic effects of secondarymetabolites will vary with the metabolite. For example, amatoxins killcells by inhibiting the function of the major cellular RNA polymerase,RNA polymerase II, in eucaryotic cells. Mutant forms of RNA polymeraseII resistant to the effects of alpha-amanitin have been described(Bartolomei et al. (1988), Mol. Cell. Biol. 8: 330-9; Chen et al.(1993), Mol. Cell. Biol. 13: 4214-22). Similarly, mutations affectingHMG CoA reductase, the target enzyme for the secondary metabolitelovastatin, have been identified. Increased levels of HMG CoA Reductasecan also cause resistance to lovastatin (Ravid et al. (1999), J. Biol.Chem. 274: 29341-51; Lum et al. (1996), Yeast 12: 1107-24). Taxol(paclitaxel), causes lethality by increasing microtubule stability, thuspreventing exit from mitosis. Dominant mutations affecting β-tubulinthat confer resistance to taxol have been characterized (for example,see Gonzalez et aL (1999), J. Biol. Chem. 274:23875-82) and could proveto be useful to confer resistance to this toxic metabolite in productionstrains. The pneumocandin and echinocandin families of metabolites arefungal secondary metabolites that inhibit the enzyme 1,3-β-D-glucansynthase. Dominant mutations in the C. albicans glucan synthase gene,FKSI, have been shown to confer resistance to candins (Douglas et al.(1997), Antinzicrob. Agents Chemother. 41: 2471-9). Glucan synthasemutations such as these could be used to generate fungal productionstrains with increased resistance to the candin class of antifungals. S.cerevisiae mutants resistant to the growth-inhibitory effects of thefungal secondary metabolite cyclosporin A have also been described(Cardenas et al. (1995), EMBO J. 14: 2772-83). These mutants were shownto harbor mutations in CNA1, the gene encoding the catalytic subunit ofthe heterodimeric calcium-calmodulin dependent phosphatase, calcineurinA. Fumagillin, an antiangiogenic agent, binds to and inhibits theN-terminal aminopeptidases in a wide variety of both procaryotes andeucaryotes (Sin et al. (1997), Proc. Natl. Acad. Sci. USA 94: 6099-103,Lowther et al. (1998), Proc. Natl. Acad Sci. USA 95: 12153-7). Mutationsin this enzyme that block fumagillin binding and/or inhibitory activitycould well prove useful in enhancing the resistance of fungal productionstrains to the growth inhibitory effects of this secondary metabolite.“Modulating the expression of a ZBC gene” is as used for the thirdaspect of the invention. In certain embodiments of the methods accordingto this aspect of the invention, the modulation is over-expression ofthe gene. “Over-expression of the gene” is as used before. In certainembodiments of the methods according to this aspect of the invention,the modulation is expression of a dominant mutation of the gene. Theterm “dominant mutation” is as used before. Preferred dominant mutationsaccording to this aspect of the invention are as used before. In certainembodiments of the methods according to this aspect of the invention,the modulation is conditional expression of the gene. The term“conditional expression” of a gene is as used before.

[0077] In the methods according to this aspect of the invention, theterm “secondary metabolite” is as used previously and preferredsecondary metabolites include, without limitation, those discussedpreviously. In certain embodiments of the methods according to thisaspect of the invention, the methods further comprise purifying thesecondary metabolite from a culture of the fungus. The term “purifying”is as used before.

[0078] In an eighth aspect, the invention provides methods for improvingproduction of a secondary metabolite in a fungus by modulating theexpression of one or more genes, the method comprising modulating theexpression of a ZBC gene or gene variant that does not normally modulatethe expression of such gene or genes.

[0079] In a ninth aspect, the invention provides genetically modifiedfungi, wherein the genetically modified fungi have an ability to producesecondary metabolites and the ability of the genetically modified fungusto produce secondary metabolites has been improved by any of the methodsaccording to the invention.

[0080] In a tenth aspect, the invention provides a method for making asecondary metabolite, the method comprising culturing a geneticallymodified fungus according to the invention under conditions suitable forthe production of secondary metabolites.

[0081] Nine ZBC genes (and derivatives thereof) have been tested to datefor effects on either lovastatin yield in A. terreus or penicillinproduction in P. chrysogenum, and in some cases both. Of these genes(lovE, lovU, An 13, At 18, CAT8, SIP4, LYS14, tamA, and YAF1) three ofthe nine (lovU, At18 and LYS14), or 33%, have demonstrable positiveeffects on metabolite production. Details of the lovU, At18 and LYS14results are described in the examples presented below.

[0082] The following examples illustrate some preferred modes ofpracticing the present invention, but are not intended to limit thescope of the claimed invention. Alternative materials and methods may beutilized to obtain similar results.

EXAMPLE 1

[0083] Construction of an Expression Vector for the At 18 Gene from A.terreus.

[0084] To test whether a ZBC gene that is not encoded within thebiosynthetic cluster for the production of a specific metabolite canregulate the biosynthesis of that specific metabolite, the ZBC-encodingAt18 gene from A. terreus was tested for effects on lovastatinproduction. To over-express At 18 (SEQ ID NO 51) in A. terreus, At18 wasamplified with oligonucleotides MO 1715 (SEQ ID NO 1) and MO 1716 (SEQID NO 2) using Turbo Pfu DNA Polymerase and a cDNA clone of At18 as atemplate under standard conditions for polymerase chain reaction (PCR).A GATEWAY Cloning Technology (Invitrogen Corp., Carlsbad, CA) entryvector was produced from the resultant PCR product and the GATEWAYpDONR206 entry plasmid according to manufacturer's instructions. Theresultant vector, MB 1754, was then reacted in a with plasmid MB 1419 toform an expression vector according to manufacturer's instructions.MB1419 is derived from pLXZ161, a vector derived from pBC-phleo (Silar(1995), Fungal Genetics Newsletter 42: 73) that carries a phleomycinresistance cassette for selection of transformants in A. terreus, aswell as a polylinker located between the A. nidulans PGK promoter andthe A. nidulans trpc terminator. pLXZ161 is constructed as follows:First, the A. nidulans trpC terminator is amplified from genomic DNA bythe PCR using Turbo Pfu Polymerase as described by the manufacturer(Stratagene, La Jolla, Calif.). Primers used in this reaction are TRPC-1(SEQ ID NO 3) and TRPC-2 (SEQ ID NO 4). The resultant product isdigested with the restriction enzymes SacII and NotI, purified byagarose gel electrophoresis, and cloned into SacII/NotI-digestedpBC-phleo DNA, to generate pLXZ 116. Second, the A. nidulans PGKpromoter is amplified from A. nidulans genomic DNA by PCR using primersPGK1-1 (SEQ ID NO 5) and PGK1-2 (SEQ ID NO 6), Turbo Pfu Polymerase, andthe reaction conditions as described above. The resultant product isdigested with ApaI and ClaI and cloned into ApaIClaI-digested pLXZ 116,to generate pLXZ161. To produce MB 1419, the ccdB (death gene) cassettefrom pEZC7201 (hnvitrogen Corp., Carlsbad, Calif.) was amplified by PCRusing oligos MO511 (SEQ ID NO 7) and MO512 (SEQ ID NO 8), digested withClaI and NotI, and cloned into NotI/ClaI-digested pLXZ161. Thisgenerated an expression vector in which the death gene cassette residesbetween the A. nidulans PGK promoter and the A. nidulans trpC terminatorof pLXZ161. The reactions using this vector allow configuration of anygene in an entry clone to be expressed under the control of the A.nidulans PGK promoter (see GATEWAY Cloning Technology manual, InvitrogenCorp., Carlsbad, Calif.). The fungal selectable marker contained on thisplasmid is ble, which confers resistance to phleomycin. Reaction ofMB1754 with MB1419 yielded a clone (MB 1970) which is configured toexpress At 18 under control of the A. nidulans PGK promoter, with theterminator region from the A. nidulans trpC gene acting as atranscriptional terminator.

EXAMPLE 2

[0085] Transformation of A. terreus with a ZBC Gene

[0086] For transformation of A. terreus, spores were first generated byculture of strain ATCC#20542 on petri plates containing potato dextroseagar (PDA, Becton Dickinson & Co., Sparks, Mo.) at 30° C. for 3-6 days.Spores were removed from PDA either by resuspension in sterile water orTween-80 (0.1%) or by scraping directly from the plate using a sterilespatula. Yeast extract sucrose (YES ) medium (2% yeast extract, 6%sucrose) was inoculated to a density of 1-5×10⁶spores per ml andincubated with shaking in an Erlenmeyer flask at 26-30° C. for 12-16 hr(250 rpm). Mycelia were harvested by centrifugation at 3200 rpm for 10minutes, and washed in sterile water twice. Mycelia were resuspended ina filter-sterilized solution of Novozyme 234 (Sigma, St. Louis, Mo.) at2-5 mg/ml in 1 M MgSO₄ and digested at room temperature with shaking (80rpm) for 1-2 hr. Undigested material was removed from the digest byfiltration through a rayon-polyester cloth with acrylic binder and 22-25μm pores (MIRACLOTH, Calbiochem, San Diego, Calif.). After adding 1-2volumes of STC (0.8 M sorbitol, 25 mm Tris, pH 7.5, and 25 mM CaCl₂),the protoplasts were pelleted by centrifugation at 2500 rpm. Protoplastswere washed twice in STC by centrifugation. Resulting protoplasts wereresuspended to a density of 5×10⁷ per ml in a solution of STC, SPTC (40%polyethylene glycol in STC) and DMSO in a ratio of 9:1:0.1 and frozen at−80° C. Two aliquots (100 μl each) of protoplasts were mixed with 1-5 μgof either pBCphleo or MB 1970 DNA and incubated on ice for 30 min. Analiquot of SPTC (15 μl) was added to each tube and the reaction wasincubated at room temperature for 15 minutes. An additional aliquot (500μl) was added with gentle mixing, and the reaction was incubated for anadditional 15 minutes at room temperature. The reaction was nextresuspended in 25 ml of molten regeneration medium (PDA from Sigma, StLouis, Mo.) with 0.8 M sucrose, maintained at 50° C.), and poured onto a150 mm petri plate containing 25 ml of solidified regeneration mediumplus phleomycin (60-200 μg/ml). Transformants were typically visibleafter 2-5 days of incubation at 26-30° C.

[0087]A. terreus transformants were grown on modified RPM medium (WO00/37629) containing 4% glucose, 0.3% corn steep liquor (Sigma, St.Louis, Mo.), 0.2%KNO_(3,) 0.3%KH₂PO_(4,)0.05%MgSO₄.7H₂O, 0.05% NaCl,0.05% polyglycol (Dow Chemical Co., Midland, Mich.), 0.1% trace elements(14.3 g/l ZnSO_(4.)7H₂O, 2.5 g/l CuSO_(4.)5H₂O, 0.5 g/l NiCl_(2.)6H₂O,13.8 g/l FeSO₄ 7H₂O, 8.5 g/l MnSO₄.H₂O, 3g/l citric acid.H₂O(add first),1 g/l H₃BO_(3,) 1 g/l Na₂MoO_(4,) 2.5 g/l CoCl₂ 6H₂O). The final pH wasadjusted to 6.5. Spores for the inoculum were generated by culturing onplates containing minimal medium plus phleomycin for 1 week at 27 C.Spores for shake flask inoculation were removed from plates by draggingthe tip of a sterile wooden stick approximately 1 inch across the platesurface. The tip of the stick was then dipped into the shake flaskmedium and swirled gently. Cultures were grown at 27° C., 225 RPM for5-6 days.

EXAMPLE 3

[0088] Determination of Lovastatin Production

[0089] Lovastatin is known to inhibit the enzyme HMG-CoA reductase(HMGR) which converts hydroxymethylglutaryl coenzyme A (HMGCoA) andNADPH to mevalonate and NADP. This reaction can be quantified bymeasuring the change in absorbance of NADPH. To assay lovastatinproduction, 6-histidine tagged HMGR ((His)₆HMGR) was first expressed inS. cerevisiae and purified with a nickel column. A. terreus samples werefermented as described above and 0.5 mL samples were taken at day 5-6,put in a 1 mL 96-well plate and centrifuged to remove mycelia beforeassaying. Samples were transferred to another 1 mL 96-well plate andfrozen at −80° C.

[0090] Samples were thawed and 10 μL removed and diluted 1:50 in H₂O. 10μl of this diluted broth was assayed in a reaction (200 μL total)containing 1 mM L-HMGCoA, 1 mM NADPH, 0.005 mM DTT and 5 μL (His)₆HMGR.The disappearance of absorbance at 340 nm was observed over time. Thisrepresented the disappearance of NADPH. Lovastatin inhibits thisreaction. The initial velocities were calculated for the reactionscontaining samples, adjusted for dilution and compared to reactionscontaining lovastatin standards to determine levels of metaboliteproduced by regression analysis. The amounts of lovastatin produced bysix At18 transformants and six pBC-phleo (vector) transformants weredetermined. The median value of lovastatin production by the vectortransformants was approximately 100 micrograms/mL broth whereas that ofthe At18 transformants was approximately 210 micrograms/ml broth. Theseresults demonstrate that a ZBC gene that is not encoded within thebiosynthetic gene cluster of a particular secondary metabolite cannonetheless regulate the production of that metabolite.

EXAMPLE 4

[0091] Construction of an Expression Vector for the lovU Gene from A.terreus.

[0092] To test lovU (previously designated as orf13 in A. terreus,Genbank ID 4959954) function in P. chrysogenum, lovU was amplified byPCR under standard conditions well known to those in the art, using A.terreus first strand cDNA as template. To generate first strand cDNA, A.terreus (MF22; ATCC#20542) was grown for 45 hours in Production Media(Cerelose, 4.5% (w/v), Peptonized Milk, 2.5% (w/v), Autolyzed yeast,0.25% (w/v), Polyglycol P2000, 0.25% (w/v), pH to 7.0) at 25° C. Myceliawere harvested in a 50 cc syringe plugged with sterile cotton wool usinga vacuum apparatus, washed once with sterile H₂O, and snap frozen inliquid nitrogen. Mycelia were then ground to a powder under liquidnitrogen with a mortar and pestle, and homogenized in RLC buffer (RNeasyKit; Qiagen, Inc., Valencia, Calif.) using a GLH rotor-statorhomogenizer (Omni International, Warrenton, Va.) Total RNA was purifiedusing an RNeasy Maxi column according to the instructions of themanufacturer.

[0093] The polyA+fraction of the A. terreus total RNA was isolated usingOligotex beads (Qiagen, Inc., Valencia, Calif.). Purified polyA+RNA (5μg) was used to generate complementary DNA (cDNA) using SUPERSCRIPTreverse transcriptase (Gibco BRL, Rockville, Md.) according to themanufacturer's instructions. First strand cDNA was used to amplify cDNApredicted to encode lovU using PCR. Oligos used to amplify the lovU cDNAwere MO843 (SEQ ID NO 9) and MO844 (SEQ ID NO 10). The resultant lovUPCR product was cloned using GATEWAY Cloning Technology (InvitrogenCorp., Carlsbad, Ca.) to generate the entry clone MB 1201. This vectorwas then used to generate the expression vector MB1317 which encodeslovU under control of the A. nidulans PGK promoter. The dominantselectable marker for transformation on this vector is the ble geneunder control of the A. nidulans GPD promoter, which confers resistanceto the antimicrobial agent phleomycin.

EXAMPLE 5

[0094] Transformation of P. chrysogenum Strains MF1 and MF20

[0095] MB1325 (a control plasmid expressing the ble marker gene whichcauses phleomycin resistance) and MB1317 were transformed into P.chrysogenum strains MF1 (NRRL1951) and MF20 (ATCC 11702). Transformationwas accomplished in the same manner as described above for A. terreus,except transformants were selected on 30 μg/mL phleomycin. To testlevels of penicillin produced in P. chrysogenumtransformants, a plugcontaining spores and mycelia was used as the inoculum. The medium usedwas the published P2 production medium (Lein (1986), in Overproductionof Microbial Metabolites, pp. 105-139, Vanek and Hostalek (eds.),Butterworth Heinemann, Woburn, Mass.) which contains, 30% lactose, 5Xpharmamedia cotton seed flour, ammonium sulfate, calcium carbonate,potassium phosphate, potassium sulfate, and phenoxyacetic acid, at pH 7.Flasks were incubated at 26° C. with shaking at 225 rpm.

[0096] Sampling was done after 6 days of growth. 1-1.5 ml of supernatantis placed into 96-well plates. Plates were centrifuged and supernatantstransferred to a new 96-well plate. Standard samples contained 0, 25,50, 100, 200, 300, 400, and 500 μg/mL phenoxymethylpenicillin (sodiumsalt) dissolved in 10 mM potassium phosphate (pH 7.0), and assays wereconducted as described below.

EXAMPLE 6

[0097] Determination of Penicillin Production

[0098] Fermentation broth was clarified by centrifugation for 10 min at4000 g, and 40 μL of clarified fermentation broth and penicillinstandard solutions was pipetted into individual wells of a 96-well UVcollection plate. Next, 200 μL of imidazole reagent was pipetted into a96-well filter plate (0.45 micron). The derivatization reaction ofpenicillin was initiated by vacuum filtration of imidazole reagent intoa collection plate containing the aliquoted samples and standards. Thecollection plate was placed into a 96-well plate reader at 45 degreeswhile absorbance at 325 nm was monitored over 20 minutes. A MolecularDynamics (Sunnyvale, Calif.) 96-well UV/Vis plate reader was used forall spectrophotometric detection. A 1.2 M aqueous imidazole solutioncontaining mercuric chloride at a concentration of 1 mM, pH 6.8 wasprepared as follows: 8.25 g of imidazole was dissolved in 60 mL ofwater, 10 mL of 5 M HCl was added, and then 10 ML of a solution ofmercuric chloride (0.27 g dissolved in 100 mL of water) was added. ThepH was adjusted to 6.80+/−0.05 with 5 M HCl and the volume was broughtto 100 mL with water (see, e.g., Bundgaard and Ilver (1972), Journal ofPharm. Pharmac: 24: 790-794). Results for the effect of lovU onpenicillin production in MF1 are shown in FIG. 2, and the results forthe effect of lovU in MF20 are shown in FIG. 3. These resultsdemonstrate that ZBC genes from one fungal genus can regulate abiosynthetic cluster or secondary metabolite production in a fungus froma different genus.

EXAMPLE 7

[0099] Effect of At18 on Lovastatin Productivity

[0100] The At 18 construct according to Example 1 is used to transformA. terreus according to Example 2. Lovastatin concentration isdetermined according to Example 3, except that it is measured as theamount of lovastatin per unit volume of broth per gram of dry cellweight at each 24 hour time point of the fermentation and expressed asconcentration as a function of fermentation time. Transformants withincreased productivity, relative to vector controls, have a higherconcentration of lovastatin at earlier time points.

EXAMPLE 8

[0101] Effect of At18 on Undesired Metabolites

[0102] The At 18 construct according to Example 1 is used to transformA. terreus according to Example 2. Transformants are grown in productionmedia and sampled at six and twelve days. The concentration of theundesired polyketide metabolite sulochrin is measured in samples of thewhole fermentation broth (broth plus cell mass) at various time pointsfor every sample. For each whole broth sample, the pH is adjusted to 7.7and an equal volume of methanol is added. These whole broth/methanolextracts are assessed for the concentration of sulochrin at each timepoint using HPLC analysis. Conditions for determining sulochrinconcentrations by HPLC are standard (see, e.g., Vinci et aL (1991), J.Ind. Microbiol. 8: 113-120) and involve separation on a C-8 HPLC columnusing a mobile phase of 0.1% H₃PO₄-acetonitrile (40:60, v/v) andmeasurement of absorbance by sulochrin at 238 nm (see, e.g., Schimmeland Parsons (1999), Biotechnology Techniques 13:379-384.)

EXAMPLE 9

[0103] Construction of an Expression Vector for the LYS14 Gene from S.cerevisiae.

[0104] To test S. cerevisiae LYS14 function in A. terreus, LYS14 wasamplified by PCR under standard conditions from S. cerevisiae genomiclibrary DNA using primers MO931 (SEQ ID NO 11) and MO932 (SEQ ID NO 12).The resultant 2.4 kb PCR product was cloned using GATEWAY CloningTechnology (Invitrogen Corp., Carlsbad, Calif.) to generate entry cloneMB 1567. Using methods well known to those experienced in the art, MB1567 was used to generate filamentous fungal expression plasmids MB1669,MB3130, and MB3139 which encode LYS14 under the control of,respectively, the A. nidulans PGK (MB1669 and MB3130) and A. nidulansGPD (MB3139) promoters. The dominant selectable marker fortransformation on MB 1669 is the ble gene under control of the A.nidulans GPD promoter, which confers resistance to the antimicrobialagent phleomycin. MB3130 and MB3139 contain the ble gene under controlof the A. nidulans trpC promoter. Corresponding vector-only controls forthe LYS14 expression plasmids are MB2143 (GPD-ble) and MB2941(trpC-ble).

EXAMPLE 9

[0105] Determination of Lovastatin Production.

[0106] 100 μL of broth sample was removed and diluted 1:10 into 70%H₂O-30% acetonitrile (900 μl). This mixture was spun down to pelletdebris at 13000 rpm for 5 minutes. 900 μl of this diluted broth wastransferred to a vial and the sample was analyzed by HPLC. 10 μl wereinjected into a Waters HPLC system (996 photo-diode array detector, 600E pump controller and 717 autosampler) equipped with a YMC-Pack ODScolumn (Aq-302-3,150×4.6 mm ID, S-3 μM pore size) and eluted withisocratic 40% aqueous acetic acid (0.7%)-60% acetonitrile for 8 minutes.Lovastatin was detected at 238 nm, found to have a retention time of 6.5minutes, and was quantitated using a calibration curve created from purelovastatin samples.

EXAMPLE 10

[0107] Effect of LYS14 on lovastatin production.

[0108] The LYS14 constructs according to Example 8 was used to transformA. terreus according to the method of Example 2. Lovastatinconcentration was determined according to the methods of Example 9.

[0109] Thus, A. terreus strain MF22 was transformed with MB2143 (GPD-blevector control), MB1669 (PGK-LYS14, GPD-ble), MB2941 (trpC-ble vectorcontrol), MB3130 (PGK-LYS14, trpC-ble) and MB3139 (GPD-LYS14, trpC-ble).Lovastatin concentration from the corresponding vector-only was used asa control. For example, MB2143 was the vector control for MB 1669. Themedian value and highest data point for lovastatin production of theLYS14 expression plasmids MB1669 and MB3139 was greater than that of thecorresponding vector only controls. In contrast, the median value forMB3130 (PGK-LYS14, trpC-ble) was lower than that of the correspondingvector-only control, suggesting that the different properties of theexpression vector can influence ability of the construct to integrate,the locus of integration, copy number, etc. Similar results, in whichsome vectors work better than others, have been observed for othergenes. Therefore, one of skill in the art can test more than one vectorto optimize results.

[0110] Equivalents.

[0111] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the appended claims.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040077039). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. A method for improving the production of asecondary metabolite by a fungus by increasing the yield of thesecondary metabolite produced by the fungus, the method comprising:modulation of the expression of at least one ZBC gene in a manner thatimproves the yield of the secondary metabolite; wherein, the ZBC gene isnot aflR if the secondary metabolite is aflatoxin or sterigmatocystin;and wherein the ZBC gene is not lovE if the secondary metabolite islovastatin.
 2. A method as in claim 1, wherein said ZBC gene comprises anucleic acid sequence selected from the group consisting of SEQ ID NOS13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45,47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307, andfungal homologs thereof.
 3. A method as in claim 1, wherein saidmodulation of said ZBC gene expression results from transformation ofsaid fungus with an exogenously supplied nucleic acid encoding said ZBCgene.
 4. A method as in claim 1, wherein the modulation of said ZBC geneis over-expression of the ZBC gene.
 5. A method as in claim 1, whereinthe modulation of the ZBC gene is conditional expression of the ZBCgene.
 6. A method as in claim 1, wherein the secondary metabolite is anantibacterial.
 7. A method as in claim 6, wherein the antibacterial is aβ-lactam.
 8. A method as in claim 7, wherein the β-lactam is selectedfrom the group consisting of penicillins and cephalosporins.
 9. A methodas in claim 8, wherein the penicillin is selected from the groupconsisting of isopenicillin N, 6-aminopenicillanic acid (6-APA),penicillin G, penicillin N, and penicillin V.
 10. A method as in claim8, wherein the cephalosporin is selected from the group consisting ofdeacetoxycephalosporin V (DAOC V), deacetoxycephalosporin C (DAOC),deacetylcephalosporin C (DAC), 7-aminodeacetoxycephalosporanic acid(7-ADCA), cephalosporin C,7-β-(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7ACA),7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA), and7-aminocephalosporanic acid (7ACA).
 11. A method as in claim 1, whereinthe secondary metabolite is an anti-hypercholesterolemic.
 12. A methodas in claim 11, wherein the anti-hypercholesterolemic is selected fromthe group consisting of lovastatin, mevastatin, simvastatin, andpravastatin.
 13. A method as in claim 1, wherein the secondarymetabolite is an immunosuppressant.
 14. A method as in claim 13 whereinthe immunosuppressant is selected from the group consisting of membersof the cyclosporin family and beauverolide L.
 15. A method as in claim14 wherein the cyclosporin is selected from the group consisting ofcyclosporin A and cyclosporin C.
 16. A method as in claim 1, wherein thesecondary metabolite is an ergot alkaloid.
 17. A method as in claim 16,wherein the ergot alkaloid is selected from the group consisting ofclavine alkaloids, lysergic acids, lysergic acid amides, ergot peptidealkaloids, ergotamine, ergosine, ergocristine, ergocryptine,ergocornine, ergotaminine, ergosinine, ergocristinine, ergocryptinine,ergocorninine, ergonovine, ergometrinine, and ergoclavine.
 18. A methodas in claim 1, wherein the secondary metabolite is an inhibitor ofangiogenesis.
 19. A method as in claim 18, wherein the inhibitor ofangiogenesis is selected from the group consisting of fumagillin andovalicin.
 20. A method as in claim 1, wherein the secondary metaboliteis a glucan synthase inhibitor.
 21. A method as in claim 20, wherein theglucan synthase inhibitor is selected from the group consisting ofechinocandin B, pneumocandin B, aculeacin A, and papulacandin.
 22. Amethod as in claim 1, wherein the secondary metabolite is a member ofthe gliotoxin family of compounds.
 23. A method as in claim 22, whereinthe member is selected from gliotoxin and aspirochlorine.
 24. A methodas in claim 1, wherein the secondary metabolite is a fungal toxin.
 25. Amethod as in claim 24, wherein the fungal toxin is selected from thegroup consisting of aflatoxins, patulin, zearalenone, cytochalasin,griseofulvin, ergochrome, cercosporin, marticin, xanthocillin,coumarins, tricothecenes, fusidanes, sesterpenes, amatoxins, malforminA, phallotoxins, pentoxin, HC toxin, psilocybin, bufotenine, lysergicacid, sporodesmin, pulcheriminic acid, sordarins, fumonisins, ochratoxinA, and fusaric acid.
 26. A method as in claim 1, wherein the secondarymetabolite is a modulator of cell surface receptor signaling.
 27. Amethod as in claim 26, wherein the modulator of cell surface receptorsignaling is selected from the group consisting of the insulin receptoragonist L-783,281 and the cholecystokinin receptor antagonistasperlicin.
 28. A method as in claim 1, wherein the secondary metaboliteis a plant growth regulator.
 29. A method as in claim 28, wherein theplant growth regulator is selected from the group consisting ofcytokinin, auxin, gibberellin, abscisic acid, and ethylene.
 30. A methodas in claim 1, wherein the secondary metabolite is a pigment.
 31. Amethod as in claim 30 wherein the pigment is selected from the groupconsisting of melanins and carotenoids.
 32. A method as in claim 1,wherein the secondary metabolite is an insecticide.
 33. A method as inclaim 32, wherein the insecticide is nodulisporic acid.
 34. A method asin claim 1, wherein the secondary metabolite is an anti-neoplasticcompound.
 35. A method as in claim 34, wherein the antineoplasticcompound is selected from taxol (paclitaxel) and related taxoids.
 36. Amethod for improving production of a secondary metabolite by a fungus byincreasing productivity of the secondary metabolite by the fungus, themethod comprising modulating the expression of a ZBC gene or genevariant in a manner that improves the productivity of the secondarymetabolite, provided however that when the secondary metabolite isaflatoxin, then the ZBC gene is not afiR, and when the secondarymetabolite is lovastatin, then the ZBC gene is not lovE.
 37. The methodaccording to claim 36, wherein one or more ZBC genes are selected fromthe group consisting of the genes corresponding to SEQ ID NOS 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307, and fungalhomologs thereof.
 38. The method according to claim 36, wherein themodulation of the ZBC gene results from it being exogenously supplied tothe fungus.
 39. The method according to claim 36, wherein the modulationof the ZBC gene is over-expression of the ZBC gene.
 40. The methodaccording to claim 36, wherein the modulation of the ZBC gene isconditional expression of the ZBC gene.
 41. The method according toclaim 36, wherein the secondary metabolite is an antibacterial.
 42. Themethod according to claim 41, wherein the antibacterial is a β-lactam.43. The method according to claim 42, wherein the β-lactam is selectedfrom the group consisting of penicillins and cephalosporins.
 44. Themethod according to claim 43, wherein the penicillin is selected fromthe group consisting of isopenicillin N, 6-aminopenicillanic acid(6-APA), penicillin G, penicillin N, and penicillin V.
 45. The methodaccording to claim 43, wherein the cephalosporin is selected from thegroup consisting of deacetoxycephalosporin V (DAOC V),deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC),7-aminodeacetoxycephalosporanic acid (7-ADCA), cephalosporin C,7-β-(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7ACA),7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA), and7-aminocephalosporanic acid (7ACA).
 46. The method according to claim36, wherein the secondary metabolite is an anti-hypercholesterolemic.47. The method according to claim 46, wherein theanti-hypercholesterolemic is selected from the group consisting oflovastatin, mevastatin, simvastatin, and pravastatin.
 48. The methodaccording to claim 36, wherein the secondary metabolite is animmunosuppressant.
 49. The method according to claim 48 wherein theimmunosuppressant is selected from the group consisting of members ofthe cyclosporin family and beauverolide L.
 50. The method according toclaim 49 wherein the cyclosporin is selected from the group consistingof cyclosporin A and cyclosporin C.
 51. The method according to claim36, wherein the secondary metabolite is an ergot alkaloid.
 52. Themethod according to claim 51, wherein the ergot alkaloid is selectedfrom the group consisting of clavine alkaloids, lysergic acids, lysergicacid amides, ergot peptide alkaloids, ergotamine, ergosine,ergocristine, ergocryptine, ergocornine, ergotaminine, ergosinine,ergocristinine, ergocryptinine, ergocorinine, ergonovine, ergometrinine,and ergoclavine.
 53. The method according to claim 36, wherein thesecondary metabolite is an inhibitor of angiogenesis.
 54. The methodaccording to claim 53, wherein the inhibitor of angiogenesis is selectedfrom the group consisting of fumagillin and ovalicin.
 55. The methodaccording to claim 36, wherein the secondary metabolite is a glucansynthase inhibitor.
 56. The method according to claim 55, wherein theglucan synthase inhibitor is selected from the group consisting ofechinocandin B, pneumocandin B, aculeacin A, and papulacandin.
 57. Themethod according to claim 36, wherein the secondary metabolite is amember of the gliotoxin family of compounds.
 58. The method according toclaim 57, wherein the member is selected from gliotoxin andaspirochlorine.
 59. The method according to claim 36, wherein thesecondary metabolite is a fungal toxin.
 60. The method according toclaim 59, wherein the fungal toxin is selected from the group consistingof aflatoxins, patulin, zearalenone, cytochalasin, griseofulvin,ergochrome, cercosporin, marticin, xanthocillin, coumarins,tricothecenes, fusidanes, sesterpenes, amatoxins, malformin A,phallotoxins, pentoxin, HC toxin, psilocybin, bufotenine, lysergic acid,sporodesmin, pulcheriminic acid, sordarins, fumonisins, ochratoxin A,and fusaric acid.
 61. The method according to claim 36, wherein thesecondary metabolite is a modulator of cell surface receptor signaling.62. The method according to claim 61, wherein the modulator of cellsurface receptor signaling is selected from the group consisting of theinsulin receptor agonist L-783,281 and the cholecystokinin receptorantagonist asperlicin.
 63. The method according to claim 36, wherein thesecondary metabolite is a plant growth regulator.
 64. The methodaccording to claim 63, wherein the plant growth regulator is selectedfrom the group consisting of cytokinin, auxin, gibberellin, abscisicacid, and ethylene.
 65. The method according to claim 36, wherein thesecondary metabolite is a pigment.
 66. The method according to claim 65,wherein the pigment is selected from a modified fungus.
 67. The methodaccording to claim 30 wherein the pigment is selected from the groupconsisting of melanins and carotenoids.
 68. The method according toclaim 1, wherein the secondary metabolite is an insecticide.
 69. Themethod according to claim 32, wherein the insecticide is nodulisporicacid.
 70. The method according to claim 1, wherein the secondarymetabolite is an anti-neoplastic compound.
 71. The method according toclaim 34, wherein the antineoplastic compound is selected from taxol(paclitaxel) and related taxoids.
 72. A method for improving productionof a secondary metabolite in a fungus by decreasing production of sideproducts or non-desired secondary metabolites, the method comprisingmodulating the expression of a ZBC gene or gene variant in a manner thatdecreases production of side products or non-desired secondarymetabolites.
 73. A method for improving production of a secondarymetabolite in a fungus by altering the characteristics of the fungus ina manner that is beneficial to the production of the secondarymetabolite, the method comprising modulating the expression of a ZBCgene or gene variant in a manner that alters the characteristics of thefungus.
 74. A method for improving production of a secondary metabolitein a fungus by causing conditional lysis of the fungus, the methodcomprising modulating the expression of a ZBC gene or gene variant in amanner that causes conditional lysis.
 75. A method for improvingproduction of a secondary metabolite in a fungus by increasing theresistance of the fungus to the deleterious effects of exposure to asecondary metabolite made by the same organism, the method comprisingmodulating the expression of a ZBC gene or gene variant in a manner thatincreases resistance to the deleterious effects of exposure to thesecondary metabolite.
 76. A method for improving production of asecondary metabolite in a fungus by modulating the expression of one ormore genes, the method comprising modulating the expression of a ZBCgene or gene variant that does not normally modulate the expression ofsuch gene or genes.
 77. A genetically modified fungus, wherein thegenetically modified fungus has an ability to produce secondarymetabolites and the ability of the genetically modified fungus toproduce secondary metabolites has been improved by the method accordingto any of claims 1-76 or 78-83.
 78. A method for making a secondarymetabolite, the method comprising culturing a genetically modifiedfungus according to claim 77 under conditions suitable for theproduction of secondary metabolites.
 79. The method according to any ofclaims 1-35, wherein the ZBC gene is from an organism in which it is notpresent within a biosynthetic cluster of a secondary metabolite.
 80. Themethod according to any of claims 1-35, wherein the ZBC gene is from anorganism other than the fungus in which the secondary metabolite isproduced.
 81. The method according to any of claims 1-35, wherein theZBC gene in its native locus regulates a different secondary metabolitethan the desired secondary metabolite produced by the fungus.
 82. Themethod according to any of claims 1-35, wherein the ZBC gene in itsnative locus does not regulate secondary metabolism.
 83. The methodaccording to claim 1, wherein the gene variant is a dominant mutation.84. The method according to claim 36, wherein the gene variant is adominant mutation.